Since the inception of this project, a number of advances have been made. First and foremost, using animal models, this project was the first to demonstrate that inherited susceptibility genes exist that predispose individuals to the development of metastatic disease. Subsequently, we demonstrated that polymorphisms in the human orthologs of these genes were also associated with distant metastasis-free survival, demonstrating an important role for these genes in human disease. Further, analysis of the human data has shown that multiple susceptibility mechanisms exist for human breast cancer metastasis since polymorphisms that predispose women with estrogen receptor positive breast cancer were not associated with metastasis in estrogen receptor negative tumors and vice versa. Analysis of the TCGA data and breast cancer susceptibility data sets suggest the genes discovered in the metastasis susceptibility screens are not associated with predisposition for breast cancer nor are they frequently somatically mutated. These genes therefore represent a novel class of genes that are associated with efficiency with which tumor cells and the collaborating tumor-associated stroma proceed through the metastatic cascade. The past year has seen a number of new advances. First, three additional inherited metastasis susceptibility genes were published (mir290, Arid4b, and Cadm1) for a total of 11 genes reported to date. To the best of our knowledge, the mir290 report is the first example that inherited variation in microRNA expression plays an important role in metastatic disease. Arid4b, a gene associated with imprinting and heterochromatin formation, is a member of the SIN3 histone deacetylase complex, which has previously been implicated in breast cancer metastasis. Intriguingly and unexpectedly, Arid4b was found to be a target of the mir290 cluster indicating that the unbiased genetic screens were at least, in part, interrogating common molecular mechanisms. Cadm1 is an adherins junction protein and evidence generated in the laboratory suggests that the anti-metastatic role of Cadm1 is at least partially due to an immune editing role, in addition to potential roles in cell-cell adhesion. Current efforts continue to build upon our past efforts and experiences. Molecular studies investigating the mechanistic role of the metastasis susceptibility genes has revealed that many of them are associated with a common complex on the inner face of the nuclear membrane. The metastasis susceptibility proteins (BRD4, isoform 2, SIPA1, RRP1B) interact with the LINC complex which connects the inner nuclear membrane to the cytoskeleton. This raises the intriguing possibility that mechanotransduction, the mechanical transmission of extracellular signals through cell adhesion molecules across the cytoskeleton and into the nucleus, may play an important role in metastatic disease. Inherited differences in the ability to transmit mechanical signals might also provide an explanation for the association between expression of various extracellular matrix molecules and breast cancer progression. Modeling in an ex vivo lung culture system, developed in the Khanna laboratory, has demonstrated that the extracellular milieu from metastasis susceptible animals promotes metastatic growth consistent with this possibility. Our laboratory continued to exploit the latest genetic tools for further identification of additional metastasis-associated loci. As new reagents have become available to permit high density genotyping, efforts have shifted away from using common laboratory strains toward strains with greater polymorphic content. Currently efforts are focused on screens utilizing the MOLF or Diversity Outbred mice. Preliminary analysis of these screens indicate that use of these mouse resources have expanded our ability to identify metastasis-associated genes. In addition to the genes primarily associated with estrogen receptor positive breast cancer found inthe original experiments, the new genetic screens have identified loci specifically associated with metastasis in estrogen negative breast cancer. An even more exciting discovery has been the identification of a locus that alters the metastatic target organ. Animals carrying this locus have a high probability to develop kidney rather than pulmonary metastases. To the best of our knowledge, this is the first demonstration that inherited factors play an important role in metastatic organ tropism. Furthermore, the locus in question contains a single gene that contains a kinase domain and therefore may be targeted by small molecule agents. In addition to this locus, numerous loci associated with tumor growth and metastatic disease have been identified. Efforts to validate these additional loci as well as identify the molecular and cellular pathways implicated are currently ongoing.